Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis

Quinn, C. P. ; Singh, Y. ; Klimpel, K. R. ; Leppla, S. H. (1991) Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis The Journal of Biological Chemistry, 266 (30). pp. 20124-20130. ISSN 0021-9258

Full text not available from this repository.

Official URL:


Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.

Item Type:Article
Source:Copyright of this article belongs to The American Society for Biochemistry and Molecular Biology.
ID Code:74253
Deposited On:09 Dec 2011 05:33
Last Modified:09 Dec 2011 05:33

Repository Staff Only: item control page